2/12/16
BIOLOGY 207 - Dr.Locke
Lecture#21 - Physical Mapping of Genes, Chromosomes,
and Genomes (Basics of Genome Projects)
Required readings and problems:
Reading: Open Genetics, Chapter 8
Problems: Chapter 8
Optional
Griffiths (2008) 9th Ed. Readings: pp 715-720
Problems: 9th Ed. Ch. 20: 3, 4, 7, 10
Campbell (2008) 8th Ed. Readings: Concept 20.1, 20.2
Concepts:
How are physical maps constructed?
1. A Physical map of a chromosome, or chromosome segment, represents the
nucleotide sequences and is often presented as a linear map of the locations of
restriction enzyme cut sites (restriction map).
2. Restriction maps of small regions of cloned DNA can be joined together to span
larger regions or whole chromosomes.
3. Genetic and physical maps have the same order but are not always directly
proportional to one another.
Biol207 Dr. Locke’s section
Lecture#21
Fall'11
page 1
2/12/16
Physical mapping - DNA Structure of a Chromosome
Chromosomes are:
1) Single Duplex of DNA
2) Linear or circular
3) Composed of a relatively constant sequence of nucleotides
- vary from one homologue to another,
How do we describe chromosomes ?
Cytogenetic map - centromere, telomeres, arms, bands
Genetic map - distance in cM - recombinant frequency
Physical Map - DNA sequence, restriction sites
Biol207 Dr. Locke’s section
Lecture#21
Fall'11
page 2
2/12/16
Physical Map Construction:
Example: Simple organism:
Bacteriophage lambda () - like T4 but smaller - 1/3 size)
Genome is single linear duplex ~48,000 bp in length;
We know the sequence of 1 strain (wildtype - but not really).
Reference for other strains, mutant strains
 -> identify the differences -> determine abnormal vs normal

Restriction Mapping
Restriction enzyme: a variety of enzymes that recognize a specific DNA sequence and
cleave the duplex at that site.
Procedure
1) Biochemically purify Lambda virus particles: DNA + Protein coat.
2) Extract the DNA - each DNA molecule will be identical in sequence
3) Digest the DNA with restriction Enzymes - series used, combinations, too.
4) Separate DNA on an agarose gel and analyse the fragments to determine the size
of each fragment produced.
5) Results are the construction of a restriction map.
Biol207 Dr. Locke’s section
Lecture#21
Fall'11
page 3
2/12/16
Digests:
C
C/K
Single Double Single
CvnI CvnI/KpnI KpnI
29.9
26.7
17.0
17.0
14.2
14.2
8.2
7.6
7.6
1.5
1.5
Cvn I
CC TNAGG
Kpn I
GGTAC C
Example of a simple restriction map:
Cvn
________26.7___________|___7.6___|____14.2____
Cvn/Kpn __________|_____|_8.2__|_________|___________
Kpn
___17.0___|_1.5_|___________29.9_____________
If many more are used.-> Figure of lambda map.
Biol207 Dr. Locke’s section
Lecture#21
Fall'11
page 4
K
M
2/12/16
Important Points:
1. Each DNA sequence can be represented by its series of restriction endonuclease
sites.
2. Each site locates the presence of a specific short stretch of base pairs (recognition
site) 4, 6, 8 bp
3. The more sites that are mapped the higher the resolution of the restriction map.
4. The ultimate physical map is the nucleotide sequence.
Why do Restriction Mapping?
1. Quick method to characterize a DNA.
-> Can tell if it is "similar" or different from other DNA, based on the restriction map.
2. Restriction sites offer positions for convenient manipulation of the DNA.
-> Cut out restriction fragment with the gene of interest and analyse it further
- eg. sequence it; use it as a probe.
Biol207 Dr. Locke’s section
Lecture#21
Fall'11
page 5
2/12/16
Physical Mapping Large Regions of DNA
Lambda DNA is only 48,000 (48 Kb). Most chromosomes are many Mb. Currently the
only way to construct physical maps of large regions is through the joining of
smaller regions.
Each smaller region is mapped, using a clone of that DNA.
Clone: virus or plasmid DNA,
-> can replicate in a host (usually bacteria E. coli),
-> into which a segment of DNA has been inserted (recombinant DNA techniques)
Contig construction:
Contig: contiguous assembly of clones
Biol207 Dr. Locke’s section
Lecture#21
Fall'11
page 6
2/12/16
Contigs can be expanded to span a whole chromosome.
Example: YEAST Chromosome #3
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Overlapping clones used to span 315 Kb of chromosome 3.
Restriction map not shown.
Sequence has been determined for the whole chromosome.
Many genes, or open reading frames (ORF), total is 182.
Only 34 identified by classical genetic techniques The function of the remaining genes is unknown.
Current research is working towards the identification and characterization of the
genes.
Having the sequence ≠ Understanding the sequence
Biol207 Dr. Locke’s section
Lecture#21
Fall'11
page 7